Alkaline phosphatase is metalloenzyme that catalyzes the hydrolysis of phosphate monoesters. The enzyme is widely distributed in nature and can be found in mammalian and bacterial systems. The exact physiological function of the enzyme is unknown, but it is thought to be involved in the transport of inorganic phosphate and in bone mineralization. This study will explore the structural and functional roles of active-site residues in bacterial alkaline phosphatase. Site-directed mutagenesis will be used to alter amino acid residues that are involved in complexing the active-site metals. Initial studies will focus on mutations at two sites; Asp-51 and Asp-153. These residues will be replaced by Ala, Asn, and His. Steady- state spectrophotometric kinetic studies and NMR studies with the mutant enzymes should provide insight into the importance of the metal centers in the catalytic process. A long term goal of this project is to see if the activity of the bacterial enzyme can be increased to that found in the mammalian form. Modification of the metal-ligand structure may provide a way to achieve this "forced evolution." Increasing our knowledge of these structure-function relationship will contribute to the basic understanding of catalysis and contribute to efforts to design enzyme inhibitors and synthetic enzymes.